Many diseases occurring in humans and animals can be detected by the presence of foreign substances, particularly in the blood, which are specifically associated with the disease or condition. Tests for antigens or other such substances produced as a result of such diseases show great promise as a diagnostic tool for the early detection and treatment of the particular disease that produced the antigen or other substance. Procedures for the detection of such substances must be reliable, reproducible, and sensitive in order to constitute a practical diagnostic procedure for health care providers. In addition, any such procedure should be able to be carried out by persons of ordinary skill and training in laboratory procedure, and should be relatively fast and inexpensive. Preferably, such procedures should be readily adaptable to instrumentation and automation, as required if such procedures are to be carried out on a large scale.
For example, in the treatment of the various malignancies that afflict humans and animals, referred to generally as cancer, it is recognized that early detection is a key to effective treatment, especially as many therapeutic procedures are effective only in relatively early stages of the disease. In fact, virtually all known cancer treatments are not only more effective, but safer, when administered in early stages of cancer. Far too many cases of cancer are only discovered too late for effective treatment.
Accordingly, there is a great need for rapid, easy-to-perform, and reliable tests which can diagnose cancer at early stages. In this connection new tests and procedures are being developed to effect early diagnosis of the cancer.
We have developed and reported one such test for the early detection of cancer in L. Cercek, B. Cercek, and C.I.V. Franklin, "Biophysical Differentiation Between Lymphocytes from Healthy Donors, Patients with Malignant Diseases and Other Disorders," Brit. J. Cancer 29, 245-352 1974) and L. Cercek and B. Cercek, "Application of the Phenomenon of Changes in the Structuredness of Cytoplasmic Matrix (SCM) in the Diagnosis of Malignant Disorder: a Review," Europ. J. Cancer 13, 903-915 (1977), which are incorporated herein by this reference.
Our basic SCM test includes the steps of:
(1) challenging a selected subpopulation of lymphocytes from a donor with a challenging agent such as a mitogen or an antigen associated with a condition or disease, such as cancer; and PA1 (2) determining the change in structuredness of the cytoplasmic matrix (SCM) of the challenged lymphocytes, typically using fluorescence polarization. PA1 (a) I.sub.P1, which is the measured fluorescence intensity in the first plane at .lambda..sub.1 ; and PA1 (b) I.sub.T1, which is the measured fluorescence intensity in the second plane at .lambda..sub.1. PA1 (i) I.sub.P2, which is the measured fluorescence intensity in the first plane at .lambda..sub.2 ; and PA1 (a) I.sub.tot1F is the total fluorescence emission intensity from the fluorescing material at .lambda..sub.1 ; PA1 (c) I.sub.tot1B is the total fluorescence emission intensity from the background material at .lambda..sub.1 ; and PA1 (d) I.sub.tot2B is the total fluorescence emission intensity from the background material at .lambda..sub.2. This is equivalent to selecting .lambda..sub.2 such that the difference between K.sub.a and K.sub.b is maximized, where: PA1 (i) I.sub.tot.lambda. is the total intensity of the fluorescence emissions from the sample at the wavelength .lambda., with .lambda. being equal to either .lambda..sub.1 or .lambda..sub.2 ; PA1 (ii) I.sub.P.lambda. and I.sub.T.lambda. are either I.sub.P1 and I.sub.T1 or I.sub.P2 and I.sub.T2, depending upon the value of .lambda.; and PA1 (iii) G is a correction factor for the unequal transmission of the vertically and horizontally polarized fluorescence emissions through the optical system of a fluorescence measuring instrument, and is constant for any particular fluorescence measuring instrument. As measured, both I.sub.P1 and I.sub.T1 include contributions of fluorescence from both the fluorescing material and the background material. PA1 (i) the vertically and horizontally polarized fluorescence emissions from the sample, I.sub.P3 and I.sub.T3 ; PA1 (ii) the total fluorescence emission intensity from the background material, I.sub.tot3B, the fluorescing material having been removed from the sample; and PA1 (iii) the total fluorescence emission intensity from the fluorescing material, I.sub.tot3P. PA1 (1) drawing a sample of lymphocyte-containing body fluid from the body to be tested; PA1 (2) separating SCM-responding lymphocytes from the body fluid; PA1 (3) contacting the SCM-responding lymphocytes with an antigen derived from or associated with the disease or condition being tested for to stimulate the lymphocytes; PA1 (4) forming a suspension of the stimulated lymphocytes and a fluorogenic agent precursor; and PA1 (5) maintaining the stimulated lymphocytes in the suspension for sufficient time to allow penetration or the lymphocytes by the fluorogenic agent precursor and the precursor's intracellular hydrolysis to a fluorogenic compound for generating stimulated fluor-containing lymphocytes. PA1 (1) an excitation source for exciting the sample at a selected excitation wavelength; PA1 (2) a fixed polarizer transmitting to the sample only plane-polarized light, the polarizer being disposed between the light source and the sample; PA1 (3) orientation selection means for selectively transmitting plane polarized light in either a first plane parallel to the plane of polarization of the exciting light or a second plane transverse to the first plane, the orientation selection means being disposed in the light path of the fluorescence emitted by the sample; PA1 (4) wavelength selection means for selecting either the primary wavelength, .lambda..sub.1 or the secondary wavelength, .lambda..sub.2, or subsequent fluorescence emission intensity measurements, the wavelength selection means being disposed in the path of the light emitted from the wavelength selection means; PA1 (5) measuring means for measuring the intensities of the components of the emitted fluorescence polarized in the first and second planes at the wavelength selected by the wavelength selection means; and PA1 (6) calculation means for calculating the net polarization value, P, of the fluorescing material in the sample from the measured intensities. PA1 (1) an excitation source; PA1 (2) a polarizer disposed between the excitation source and the sample; PA1 (3) a first fixed orientation selection means for selectively transmitting plane-polarized light only in a first plane, the first fixed orientation selection means being disposed in the light path of the fluorescence emitted by the sample; PA1 (4) a second fixed orientation selection means for selectively transmitting plane-polarized light only in a second plane transverse to the first plane, the second fixed orientation selection means being disposed in the light path of the fluorescence emitted by the sample; PA1 (5) a first wavelength selection means for selecting only a primary wavelength, .lambda..sub.1, for subsequent fluorescence emission intensity measurements, the first wavelength selection means being disposed in the path of the light emitted from the first fixed orientation selection means; PA1 (6) a second wavelength selection means for selecting only a secondary wavelength, .lambda..sub.2, for subsequent fluorescence intensity measurements, the second wavelength selection means being disposed in the path of the light emitted from the second fixed orientation selection means; PA1 (7) measuring means for measuring the intensities of the components of the emitted fluorescence polarized in the first and second planes at the wavelengths selected by the first and second wavelength selection means; and PA1 (8) calculation means for calculating the net polarization value, P, of the fluorescing material in the sample from the measured intensities. PA1 (1) an excitation source; PA1 (2) a fixed polarizer; PA1 (3) orientation selection means for selectively transmitting plane polarized light in either a first plane or the second plane, the orientation selection means being movably disposed alternately in the light path of the fluorescence emitted by the sample or outside of the light path; PA1 (4) wavelength selection means; PA1 (5) positioning means interlocked with the wavelength selection means such that the positioning means positions the orientation selection means in the light path only whenever the wavelength selection means selects .lambda..sub.1 and positions the orientation selection means outside the light path whenever the wavelength selection means selects .lambda..sub.2 ; PA1 (6) a first measuring means for measuring the intensity of the components of the emitted fluorescence polarized in the first and second planes at .lambda..sub.1 whenever the orientation selection means is disposed in the light path; PA1 (7) a second measuring means for measuring I.sub.tot2, the total intensity of the fluorescence emitted from the sample at .lambda..sub.2 whenever the orientation selection means is outside of the light path; and PA1 (8) calculation means. PA1 (1) a first photodetector disposed in the exit light path of the first emission monochromator to which the light transmitted by the orientation selection means when the orientation selection means is in the light path and transmits plane polarized light in the first plane is directed; and PA1 (1) an excitation source; PA1 (2) a fixed polarizer; PA1 (3) orientation selection means for selectively transmitting plane polarized light in one of three planes: a vertical plane, a horizontal plane, and a plane oriented 54.7.degree. from the vertical, the orientation selection means disposed in the path of the fluorescence emitted by the sample; PA1 (4) wavelength selection means for selecting either .lambda..sub.1 or .lambda..sub.2 for subsequent fluorescence emission intensity measurements, the wavelength selection means interlocked with the orientation selection means so that .lambda..sub.1 is selected whenever the orientation selection means transmits light in either the vertical plane or the horizontal plane, and so that .lambda..sub.2 is selected whenever the orientation selection means transmits light in the plane oriented 54.7.degree. from the vertical; PA1 (5)measuring means for measuring the intensities of the components of the emitted fluorescence polarized in the vertical plane, the horizontal plane, and the plane oriented 54.7.degree. from the vertical, at the wavelength selected by the wavelength selection means; and PA1 (6) calculation means for determining I.sub.tot2 from I.sub.M2 and calculating P.
When applied to cancer, our SCM (structuredness of cytoplasmic matrix) test is based on the phenomenon that the internal structure of a selected subpopulation of the lymphocytes from a healthy individual is altered when challenged by a mitogen such as phytohaemagglutinin (PHA) but is not altered by other selected challenging agents, such as certain cancerassociated antigens. Contrarily, the equivalent subpopulation of lymphocytes from an individual with cancer responds oppositely. In other words the same subpopulation of lymphocytes from cancer patients does not respond in the SCM test when challenged by a mitogen, but does respond strongly to challenge by a number of cancer-associated antigens.
The change seen in SCM are believed to reflect changes in the internal structure of the lymphocyte as the lymphocyte is activated for synthesis. Similar changes can occur in living cells other than lymphocytes during the cell cycle and growth of the cells. Such changes can also be evoked by various external agents, such as ionizing radiation, mechanical forces, chemicals, growth inhibiting and stimulating agents, etc. These changes can be conveniently monitored with a specially adapted technique of fluorescein fluorescence polarization, as we have published in numerous articles, including L. Cercek and B. Cercek, "Studies on the Structuredness of Cytoplasm and Rates of Enzymatic Hydrolysis in Growing Yeast Cells. I. Changes Induced by Ionizing Radiation," Int. J. Radiat. Biol. 21, 445-453 (1972); L. Cercek and B Cercek, "Studies on the Structuredness of Cytoplasm and Rates of Enzymatic Hydrolysis in Growing Yeast Cells. II. Changes Induced by Ultra-Violet Light," Int. J. Radiat. Biol 22. 539-544 (1972); L. Cercek and B. Cercek, "Relationship Between Changes in the Structuredness of Cytoplasm and Rate Constants for the Hydrolysis of FDA in Saccharomyces cerevisiae," Biophysik 9, 109-112 (1973); L. Cercek, B. Cercek, and C. H. Ockey, "Structuredness of the Cytoplasmic Matrix and Michaelis-Menten Constants for the Hydrolysis of FDA During the Cell Cycle in Chinese Hamster Ovary Cells," Biophysik 10 187-194 (1973) B. I. Lord, L. Cercek, B. Cercek, G. P. Shah, T. M. Dexter and L. G. Lajtha, "Inhibitors of Haemopoietic Cell Proliferation: Specificity of Action Within the Haemopoietic System," Brit. J. Cancer 29 168-175 (1974); L. Cercek and B. Cercek, "Involvement of Cyclic-AMP in Changes of the Structuredness of Cytoplasmic Matrix," Radiat. & Environ. Biophys. 11, 209-212 (1974); L. Cercek, P. Milenkovic, B. Cercek, & L. G. Lajtha, "Induction of PHA Response in Mouse Bone Marrow Cells by Thymic Extracts as Studied by Changes in the Structuredness of Cytoplasmic Matrix," Immunology 29, 885-891 (1975); L. Cercek and B. Cercek, "Effects of Osmomolarity, Calcium and Magnesium Ions on the Structuredness of Cytoplasmic Matrix (SCM)," Radiat. & Environ. Biophys. 13, 9-12 (1976); L. Cercek & B. Cercek, "Changes in the Structuredness of Cytoplasmic Matrix (SCM) Induced in Mixed Lymphocyte Reactions," Radiat. & Environ. Biophys. 13, 71-74 (1976); L. Cercek, B. Cercek, & C. H. Ockey, "Fluorescein Excitation and Emission Polarization Spectra in Living Cells: Changes During the Cell Cycle," Biophys. J. 23, 395-405 (1978) L. Cercek and B. Cercek, "Involvement of Mitochondria in Changes of Fluorescein Excitation and Emission Polarization Spectra in Living Cells," Biophys. J. 28, 403-412 (1979); L. Cercek, B. Cercek, and B. I. Lord, "The Effect of Specific Growth Inhibitors on Fluorescein Fluorescence Polarization Spectra in Haemopoietic Cells," Brit. J. Cancer, 44 749-752 (1981); and L. Cercek and B. Cercek, "Effects of Ascorbate Ions on Intracellular Fluorescein Emission Polarization Spectra in Cancer and Normal Proliferating Cells," Cancer Detection and Prevention 10, 1-20 (1987), all of which are incorporated herein by this reference.
The usefulness of this SCM test for the detection of cancer has been documented in numerous articles. Articles from our laboratory include: L. Cercek, B. Cercek, and J. V. Garrett, "Biophysical Differentiation Between Normal Human and Chronic Lymphocytic Leukaemia Lymphocytes," in Lymphocyte Recognition and Effector Mechanisms (K. Lindahl-Kiessling and D. Osoba eds., New York, Academic Press, 1974), pp. 553-558; L. Cercek, B. Cercek and C. I. V. Franklin, "Biophysical Differentiation between Lymphocytes from Healthy Donors, Patients with Malignant Disease and Other Disorders," Brit. J. Cancer 29 345-352 (1974); L. Cercek and B. Cercek, "Changes in the SCM Response Ratio (RR.sub.SCM) After Surgical Removal of Malignant Tissue," Brit. J. Cancer 31, 250-251 (1975); L. Cercek and B. Cercek, "Apparent Tumour Specificity with the SCM Test," Brit. J. Cancer 31, 252-253 (1975); L. Cercek and B. Cercek, "Changes in the Structuredness of Cytoplasmic Matrix of Lymphocytes as a Diagnostic and Prognostic Test for Cancer," in Cell Biology and Tumour Immunology, Excerpta Medica International Congress Series No. 349, Proceedings of the XI International Cancer Congress, Florence, 1974 (Amsterdam, Excerpta Medica, 1974), vol. 1, pp. 318-323) L. Cercek and B. Cercek, "Application of the Phenomenon of Changes in the Structuredness of Cytoplasmic Matrix (SCM) in the Diagnosis of Malignant Disorders: a Review," Europ. J. Cancer 13, 903-915 (1977); L. Cercek and B. Cercek, "Detection of Malignant Diseases by Changes in the Structuredness of Cytoplasmic Matrix of Lymphocytes Induced by Phytohaemagglutinin and Cancer Basic Proteins," in Tumour Markers, Determination and Clinical Role: Proceedings of the Sixth Tenovus Workshop, Cardiff, April 1977 (K. Griffith, A. M. Neville, and C. G. Pierrepoint, eds., Cardiff, Alpha Omega Publishing Co., 1978), pp. 215-226; and L. Cercek and B. Cercek, "Changes in SCM-Responses of Lymphocytes in Mice After Implantation with Ehrlich Ascites Cells," Europ. J. Cancer 17, 167-171 (1981), all of which are incorporated herein by this reference.
The usefulness of the SCM test has been confirmed in articles from other laboratories, including F. Takaku, K. Yamanaka, and Y. Hashimoto, "Usefulness of the SCM Test in the Diagnosis of Gastric Cancer," Brit. J. Cancer 36, 810-813 (1977); H. Kreutzmann, T. M. Fliedner, H. J. Galla, and E. Sackmann, "FluorescencePolarization Changes in Mononuclear Blood Leucocytes After PHA Incubation: Differences in Cells from Patients with and Without Neoplasia," Brit. J. Cancer 37, 797-805 (1978); Y. Hashimoto, T. Yamanaka, and F. Takaku, "Differentiation Between Patients with Malignant Diseases and Non-Malignant Diseases or Healthy Donors by Changes of Fluorescence Polarization in the Cytoplasm of Circulating Lymphocytes," Gann 69, 145-149 (1978); J. A. V. Pritchard and W. H. Sutherland, "Lymphocyte Response to Antigen Stimulation as Measured by Fluorescence Polarization (SCM-Test)," Brit. J. Cancer 38, 339-343 (1978); J. A. V. Pritchard, J. E. Seaman, I. H. Evans, K. W. James, W. H. Sutherland, T. J. Deeley, I. J. Kerby, I. C. M. Patterson, and B. H. Davies, "Cancer-Specific Density Changes in Lymphocytes Following Stimulation with Phytohaemagglutinin," Lancet 11, 1275-1277 (Dec. 16. 1978); H. Orjasaeter, G. Jordfald, and I. Svendsen, "Response of T-Lymphocytes to Phytohaemagglutinin (PHA) and to Cancer-Tissue-Associated Antigens, Measured by the Intracellular Fluorescence Folarization Technique (SCM Test)," Brit. J. Cancer 40, 628-633 (1979); N. D. Schnuda, "Evaluation of Fluorescence Polarization of Human Blood Lymphocytes (SCM Test) in the Diagnosis of Cancer," Cancer 46, 1164-1173 (1980); J. A. V. Pritchard, W. H. Sutherland, J. E. Siddall, A. J. Bater, I. J. Kerby, T. J. Deeley, G. Griffith, R. Sinclair, B. H. Davies, A. Rimmer, & D. J. T. Webster, "A Clinical Assessment of Fluorescence Polarisation Changes in Lymphocytes Stimulated by Phytohaemagglutinin (PHA) in Malignant and Benign Disease," Europ. J. Cancer, Clin. Oncol. 18, 651-659 (1982); G. R. Hocking, J. M. Rolland, R. C. Nairn, E. Pihl, A. M. Cuthbertson, E. S. R. Hughes, and W. R. Johnson, "Lymphocyte Fluorescence Polarization Changes After Phytohaemagglutinin Stimulation in the Diagnosis of Colorectal Carcinoma," J. National Cancer Inst. 68 579-583 (1982); M. Deutsch and A. Weinreb, "Validation of the SCM-Test for the Diagnosis of Cancer," Eur. J. Cancer, Clin. Oncol. 19, 187-193 (1983); S. Chaitchik, O. Asher, M. Deutsch. and A. Weinreb, "Tumour Specificity of the SCM Test for Cancer Diagnosis," Europ. J. Cancer, Clin. Oncol. 21 1165-1170 (1985); and J. Matsumoto, T. Tenzaki and T. Ishiguro, "Clinical Evaluation of Fluorescein Polarization of Peripheral Lymphocytes (SCM Test) in the Diagnosis of Cancer," J. Japan Soc. Cancer Ther. 20, 728-734 (1985), all of which are incorporated herein by this reference.
The SCM test can be applied to detection of diseases and conditions other than cancer, such as viral and bacterial infections, determination of allergic reactions, tissue typing, and monitoring of allograft rejections based on the SCM responses in mixed lymphocyte reactions, as disclosed in the 1976 Radiation and Environmental Biophysics article by L. Cercek and B. Cercek. This extension of the SCM test is disclosed and claimed in our co-pending U.S. patent application, Ser. No. 838,264, filed Mar. 10, 1986, now abandoned, and entitled "Separation and Use of Density Specific Blood Cells," which is incorporated herein by this reference. The presence of other antigen-producing diseases and bodily conditions does not interfere with the SCM test; a patient afflicted with more than one type of antigen-producing disease can be tested for a multiplicity of such diseases simply by running separate tests using for each test an antigen derived from each separate disease or condition being tested for.
When fluorescence polarization is used to determine changes of SCM, such changes are seen as a decrease in the fluorescence polarization of the cells when polarized light is used to excite an extrinsic fluor generated intracellularly by the hydrolysis of a nonfluorescent compound which has been absorbed by the lymphocytes. The fluor typically is fluorescein and the nonfluorescent compound is typically fluorescein diacetate (FDA). The FDA serves as a fluorogenic agent precursor. An extrinsic fluor is used because the intrinsic fluorescence of cellular components is too small to give meaningful results in this test. Therefore, all references to fluorescence polarization values herein are references to fluorescence polarization values obtained with an extrinsic fluor, preferably one generated by enzymatic hydrolysis from a nonfluorogenic compound added to and absorbed by the cells.
Fluorescence polarization is a measure of intracellular rigidity; the greater the intracellular mobility, the less the measured fluorescence polarization. As seen in the SCM test, the observed decrease in fluorescence polarization is believed to result mainly from changes in the conformation of the mitochondria, the energy-producing organelles of the cell. The changes in the mitochondria are believed to result from the contractions of the cristae or inner folds of the mitochondrial membrane. The SCM reflects the forces of interaction between macromolecules and small molecules such as water molecules, ions, adenosine triphosphate, and cyclic adenosine monophosphate. Perturbations of these interactions result in changes in the SCM.
In our SCM test, the best indication of structuredness is not the absolute fluorescence polarization measured, but rather the net fluorescence polarization (P). P is determined after correction is made for: (i) intrinsic fluorescence of the medium in which the cells are suspended; (ii) extracellular fluor present whether generated by leakage of fluor from cells or non-enzymatic hydrolysis of fluorescein diacetate in the medium; and (iii) unequal transmission of the two components of polarized light in the fluorescence polarization measurement apparatus. Thus all references to fluorescence polarization below are to net fluorescence polarization, P, unless indicated otherwise. When the fluorescence polarization measurements are performed on living cells, P is the net intracellular polarization.
In our test, fluorescein is introduced into the cells by intracellular hydrolysis of the non-fluorogenic compound fluorescein diacetate which has been taken up by the lymphocytes. Then are measured the horizontally and vertically polarized components of emitted fluorescence due to excitation of the cell suspension by light from a suitable source, such as vertically polarized blue light from a xenon lamp. The intensities of the vertically and horizontally polarized fluorescence components are used to calculate P. Challenged lymphocytes from a donor afflicted with a disease or condition associated with the challenging antigen exhibit a substantial decrease of at least 10 percent in the fluorescence polarization value, P, compared to non-challenged lymphocytes from the same donor. On the other hand, challenged lymphocytes from donors not afflicted with the antigen-producing disease or condition do not exhibit a significant decrease in P after contact with the challenging antigen.
As previously stated, the calculation of P includes corrections for several factors, including background fluorescence, in order to yield meaningful fluorescence polarization values. Ideally, since the FDA or other fluorogenic agent precursor itself does not fluoresce and is only converted into a fluorescent compound such as fluorescein on intracellular hydrolysis by the lymphocytes, the background is relatively small and consists of only the fluorescence resulting from the background material.
In practice, however, compensating for background fluorescence creates serious problems with the SCM measurements. As soon as the intracellular hydrolysis of FDA to fluorescein begins, some of the fluorescein molecules produced by the hydrolysis leak out of the cell and add to background fluorescence. Additionally, FDA is susceptible to nonenzymatic or thermal hydrolysis resulting in still more fluorescein present outside of the cell and a higher background. This background steadily increases as the fluorescence polarization is measured.
In our prior work, we compensated for this extracellular fluorescence background by filtering the lymphocyte suspension. Filtration was begun about four to seven minutes after the recording of polarized fluorescence intensities had begun. The vertically and horizontally polarized components of the fluorescence emissions from the cell-free filtrate as well as the length of time of the filtration step had to be recorded. The fluorescence polarization intensity measurements performed on the lymphocyte suspension before filtering then had to be extrapolated to the time point at which the filtration step was one-half completed, and the fluorescence polarization measurements on the filtrate then subtracted from the extrapolated measurements to obtain the net intracellular vertically and horizontally polarized fluorescence intensities due to the lymphocytes themselves. This measurement process is described in our 1977 European Journal of Cancer article.
In unskilled hands, the filtration step can introduce errors and uncertainties into the SCM results. For example, delay in filtration can introduce uncertainties in the values of the extrapolated fluorescence polarization measurements, since the intensity increases with time. In addition, if excess pressure is applied to the cells during filtration, the filtration step itself can damage cell membranes, resulting in leakage of intracellular fluorescein and fluorescein diacetate-hydrolyzing enzyme into the filtrate. This leakage can cause an artificially high background measurement. Not only can the fluorescein leaked into the filtrate directly increase the background fluorescence, but the presence of fluorescein diacetate-hydrolyzing enzyme in the filtrate can convert some of the nonfluorescing fluorescein diacetate into fluorescein, further adding to the background.
Clinical consequences of an erroneously high background measurement can be serious. Because fluorescence polarization is a measure of mobility, the emissions from the free fluorescein released by rupture of the cells or created by hydrolysis in the filtrate are less polarized than that of bound fluorescein within the lymphocytes. The subtraction of this artificially high background from the vertically and horizontally polarized fluorescence intensity measurements on the suspension can lead to the erroneous conclusion that the emissions from the lymphocyte-containing suspension are more polarized than they actually are. If the filtration error occurs on a lymphocyte sample that has been stimulated with a cancer-associated antigen, the result can be a false negative test. This occurs because the apparent polarization actually measured is greater than it should be and the decrease in fluorescence polarization caused by a positive SCM response and indicative of cancer will be masked. Conversely, if the filtration error occurs on a sample of unstimulated control lymphocytes, the result can be a false positive. The apparent polarization value of the control is higher than it should be In this case, another sample of lymphocytes from a normal donor exposed to a cancer-associated antigen but not responding to that antigen gives a lower apparent polarization value if the artificially high background measurement does not occur on that sample. This lower apparent polarization value can be interpreted as indicating the presence of cancer.
There are additional disadvantages associated with the use of the filtration technique to determine the background for fluorescence polarization measurements, especially if large-scale clinical testing is intended. Considerable experience and skill are required to extrapolate the fluorescence measurements accurately and repeatedly over the time period required for filtration. The filtration process is slow, particularly if the pressures used are limited; it requires additional equipment, and is difficult to carry out reproducibly on more than a few samples at a time. Also, the filtration procedure requires rather large volumes of sample, about 3 ml. Although all of these disadvantages can be overcome when the SCM test is used for relatively small-scale laboratory studies, they present serious obstacles to large-scale clinical use of the SCM test.
Accordingly, there is a need for a fluorescence polarization measurement technique capable of compensating for background extracellular fluorescence without using the filtration step. This technique should be rapid, suitable for automation, require a small sample, and be capable of being carried out with a minimum of equipment by workers with a minimum of specialized training. Preferably a large number of samples should be able to be processed with the technique.